Phytochemical Profiling, Antioxidant and Cognitive-Enhancing Effect of Helichrysum italicum ssp. italicum (Roth) G. Don (Asteraceae)

This study aimed at the evaluation of the antioxidant and cognitive-enhancing effect of methanol–aqueous extract from Helichrysum italicum ssp. italicum aerial parts. Significant radical scavenging activity (110.33 ± 3.47 and 234.70 ± 5.21 mg TE/g for DPPH and ABTS) and reducing power (354.23 ± 17.51 and 210.24 ± 8.68 mg TE/g for CUPRAC and FRAP) were observed. The extract showed average acetylcholinesterase and low butyrylcholinesterase inhibitory potential. H. italicum extract (200 mg/kg/po) administered in combination with galantamine (3 mg/kg/po) for 12 days significantly improved the memory and learning process compared with galantamine alone in the passive avoidance test. The effect was comparable to that of Ginkgo biloba extract (100 mg/kg/po). In deep secondary metabolite annotation of the extract by UHPLC-HRMS, more than 90 hydroxybenzoic and hydroxicinnamic acid-glycosides, phenylethanoid glycosides, a series of acylquinic and caffeoylhexaric acids, methoxylated derivatives of scutellarein, quercetagetin and 6-hydroxyluteolin, and prenylated phloroglucinol-α-pyrones were reported for the first time in H. italicum. Fragmentation patterns of four subclasses of heterodimer-pyrones were proposed. In-depth profiling of the pyrones revealed 23 compounds undescribed in the literature. Pyrones and acylphloroglucinols together with acylquinic acids could account for memory improvement. The presented research advanced our knowledge of H. italicum, highlighting the species as a rich source of secondary metabolites with cognitive-enhancing potential.


Introduction
Helichrysum italicum (Roth) G. Don (curry plant, immortelle) is an aromatic perennial subshrub belonging to the family Asteraceae, subfamily Asteroideae, and tribe Gnaphalieae. The genus Helichrysum Mill. comprises approximately 600 species distributed in central Asia, India, Africa, including Madagascar, and the Mediterranean Basin [1]. The genus has a high occurrence in the Mediterranean areas of Europe. Based on the morphological features, genetic variation, and geographical distribution, Helichrysum italicum is divided into six subspecies (ssp.): italicum, microphyllum, picardii, siculum, serotinum, and tyrrhenicum [2]. The name of the genus is derived from the Greek words "helios" and "chryos", which mean "sun" and "gold", respectively. This nomenclature is due to the inflorescences of a bright yellow color typical for the taxon [3]. The common name "immortelle" is related to the everlasting flowers that retain their form and color when dried and thus are used in dry bouquets [4].
increased the fat oxidation and resting energy expenditure and induced the metabolic effects [16]. A clinical trial investigated the result of H. italicum essential oil on mental exhaustion and moderated burnout and revealed a reduction in the perceived level of mental fatigue/burnout [17].
Despite the extensive investigation of the chemical composition and biological activities, there are limited data on the ability of H. italicum extracts to improve memory and learning. Hence, the aim of this study was to analyze the secondary metabolites in H. italicum var. italicum, in the light of its antioxidant and cognitive-enhancing effect. In addition, we hypothesized that the improvement of the learning and memory processes of H. italicum methanol-water extract could be attributed to its powerful antioxidant and moderate acetylcholinesterase inhibitory activity.

Results and Discussion
A flow chart for the design of the experiment was presented in Figure 1. H. italicum aerial parts were extracted with 80% methanol and lyophilized. The dissolved lyophilized extract was first injected in UHPLC-HRMS. Data were acquired using the data-dependent acquisition mode and then converted through a MZmine 2.53 software processing. After obtaining the UHPLC-HRMS profiling, in negative and positive ion modes, extracted chromatograms and their MS/MS spectra followed. Based on the literature data, using the taxonomy as a filter and comparing it with authentic standards, dereplication/annotation of the peaks was carried out. In parallel, spectrophotometric assays were conducted to determine antioxidant and cholinesterase inhibitory potential. Finally, the cognitiveenhancing effect was determined using an in vivo passive avoidance test. and influence of H. italicum on obesity, metabolic syndrome, and type 2 diabetes mellitus. Additionally, clinical data revealed that H. italicum subsp. italicum infusion acutely increased the fat oxidation and resting energy expenditure and induced the metabolic effects [16]. A clinical trial investigated the result of H. italicum essential oil on mental exhaustion and moderated burnout and revealed a reduction in the perceived level of mental fatigue/burnout [17].
Despite the extensive investigation of the chemical composition and biological activities, there are limited data on the ability of H. italicum extracts to improve memory and learning. Hence, the aim of this study was to analyze the secondary metabolites in H. italicum var. italicum, in the light of its antioxidant and cognitive-enhancing effect. In addition, we hypothesized that the improvement of the learning and memory processes of H. italicum methanol-water extract could be attributed to its powerful antioxidant and moderate acetylcholinesterase inhibitory activity.

Results and Discussion
A flow chart for the design of the experiment was presented in Figure 1. H. italicum aerial parts were extracted with 80% methanol and lyophilized. The dissolved lyophilized extract was first injected in UHPLC-HRMS. Data were acquired using the data-dependent acquisition mode and then converted through a MZmine 2.53 software processing. After obtaining the UHPLC-HRMS profiling, in negative and positive ion modes, extracted chromatograms and their MS/MS spectra followed. Based on the literature data, using the taxonomy as a filter and comparing it with authentic standards, dereplication/annotation of the peaks was carried out. In parallel, spectrophotometric assays were conducted to determine antioxidant and cholinesterase inhibitory potential. Finally, the cognitive-enhancing effect was determined using an in vivo passive avoidance test.

UHPLC-HRMS Profiling of H. italicum Extract
Herein, a comprehensive UHPLC-HRMS analysis of H. italicum methanol-aqueous extract was performed yielding the identification/annotation of more than 160 secondary

UHPLC-HRMS Profiling of H. italicum Extract
Herein, a comprehensive UHPLC-HRMS analysis of H. italicum methanol-aqueous extract was performed yielding the identification/annotation of more than 160 secondary metabolites (Tables 1 and S1). The total ion chromatogram (TIC) in the negative ion mode of the studied extract was depicted in Figure 2.
quercetagetin, and 6-hydroxyluteolin together with flavonol-hydroxycinnamoylglycosides represent new secondary metabolites in the species. This study is the first attempt at an in-depth characterization of phloroglucinol α-pyrones by hyphenated technique LC-MS allowing for the annotation of a series of heterodimers not previously reported even in the genus Helichrysum. It is worth noting that the fragmentation patterns of four subclasses of pyrones are suggested (Tables S1 and S2, Figures 1 and S7-S11).
Within this group, 26 (caffeic acid) was the main compound in the studied extract, together with 19, 24, and 25 ( Figure S1). Although hydroxybenzoic and hydroxycinnamic acids were present in their free form, herein, a large number of phenolic acids hexosides were revealed in H. italicum for the first time. The extracted ion chromatograms of hydroxybenzoic and hydroxycinnamic acids and derivatives showed that the H. italicum For the first time, 12 phenolic acids and derivatives and coumarins, 14 acylquinic acids, 15 acylhexaricic acids, 22 flavonoid aglycones and glycosides, and 28 heterodimerpyrones were reported in H. italicum (Table 1, Figures S1-S6). To the best of our knowledge, phenylethanoid glycosides, a series of hydroxybenzoic and hydroxicinnamic acidglycosides, hydroxycinnamoyl hexoses (sugar esters), caffeoyl-hydroxydihydrocaffeoylquinic, malonyl-dicaffeoylquinic, p-coumaroyl-caffeoylquinic and tricaffeoylquinic acids, and dicaffeoylquinic acid-hexoside were reported for the first time. Within a group of acylhexaric acids, hydroxybutanyl-tricaffeoylhexaric, isobutanyl-tricaffeoylhexaric, and 2methyllbutanyl/isovaleryl-tricaffeoylhexaric acids were not previously reported in the literature. A series of methoxylated derivatives of scutellarein, quercetagetin, and 6hydroxyluteolin together with flavonol-hydroxycinnamoylglycosides represent new secondary metabolites in the species. This study is the first attempt at an in-depth characterization of phloroglucinol α-pyrones by hyphenated technique LC-MS allowing for the annotation of a series of heterodimers not previously reported even in the genus Helichrysum. It is worth noting that the fragmentation patterns of four subclasses of pyrones are suggested (Tables S1 and S2, Figures 1 and S7-S11).

Other Compounds
The known monomer pyrone micropyrone 160 with [M-H] − at m/z 251.129 (calc. for C14H19O4) was dereplicated together with italipirone and its isomer (161/162) consisting of both pyrone and a benzofurane ring [6]. A pyrone moiety was evidenced by the transition 399.144→233.0814 resulting from the loss of ethylpyrone as was observed in the heterodimer-pyrones (Table S1).     Figure 4). The aforementioned structures are consistent with hydroxyprenyl residue at C-3, as was found in Heliarzanol [8,24]. It is worth noting that the 4-hydroxypyrone core exists in two tautomeric forms. On the other hand, the rotamers arise from the intramolecular hydrogen bonds between the oxygen functions of pyrone moiety and phenolic hydroxyl groups vicinal to the methylene bridge. Thus, different rotameric and/or tautomeric forms occur [8]. In the same way, hydroxyprenyl residue was evidenced in 138 and 154 ([M-H] − at m/z 459.203), where 2-methyl-oxobutyl residue at C-1 was deduced (Table S1) (Table S1, Figure S8). This structure was consistent with the compound previously identified in H. decumbens [30].  (Table S1). Accordingly, hydroxygeranyl residue was suggested at C-3 of both pyrones.
UHPLC-HRMS analysis revealed that phloroglucinol alpha-pyrones was the major group of secondary metabolites in H. italicum extract and reach up to 38.05% of all of the 166 compounds. Acylquinic acids (29.52%) and flavonoids (13.57%) were also found in high quantities in the studied species. Among all compounds, arzanol (135) (7.33%) was the predominant metabolite in the extract, while the amount of 3,4-dicaffeoylquinic acid (51), 3,5-dicaffeoylquinic acid (52), and 1,5-dicaffeoylquinic acid were found to be 5.61%, 5.58%, and 4.58%, respectively. The structures of the main annotated metabolites in H. italicum extract are presented in Figure 5.

Antioxidant and Cholinesterase Inhibitory Activity
In the present study, H. italicum extract was tested for antioxidant and cholinesterase inhibitory potential (Table 2). DPPH and ABTS+ were used to evaluate radical scavenging

Antioxidant and Cholinesterase Inhibitory Activity
In the present study, H. italicum extract was tested for antioxidant and cholinesterase inhibitory potential (Table 2). DPPH and ABTS+ were used to evaluate radical scavenging ability, while the reduction abilities were calculated by the CUPRAC, FRAP, and phosphomolybdenum (PHMD) methods. The metal chelating method was based on the binding of transition metals by phytochemicals. Results are presented as trolox equivalents and ethylenediaminetetraacetic acid (EDTA), and H. italicum extract revealed high activity of all of the used antioxidant methods. The enzyme inhibitory properties of H. italicum extracts were examined against both AChE and BChE. The results are calculated as a Galantamine (Gal) equivalent. The studied extract showed average AChE (1.64 ± 0.09 mg GALAE/g) and low BChE inhibitory potential (0.11 ± 0.02 mg GALAE/g). A high selectivity of the enzyme inhibitory activity of H. italicum extract targeting AChE was demonstrated.

Passive Avoidance Test
In the passive avoidance test, the ability of the animals to learn the new task was assessed on the fifth day ( Figure 6). On this day, only the group treated with the combination galantamine and H. italicum extract showed a slight increase in the latency time in comparison to the control and other groups. On day 12, when the memory of the animals was evaluated, groups treated with Ginkgo biloba and with the combination galantamine and H. italicum showed a statistically significant increase (p ≤ 0.001) in the latency times compared to the control group (Figure 7). The result indicated an improvement of the memory processes after 12 days of administration of these compounds. strated.

Passive Avoidance Test
In the passive avoidance test, the ability of the animals to learn the new tas assessed on the fifth day ( Figure 6). On this day, only the group treated with the c nation galantamine and H. italicum extract showed a slight increase in the latency t comparison to the control and other groups. On day 12, when the memory of the an was evaluated, groups treated with Ginkgo biloba and with the combination galant and H. italicum showed a statistically significant increase (p ≤ 0.001) in the latency compared to the control group (Figure 7). The result indicated an improvement memory processes after 12 days of administration of these compounds.  The incidence of dementia, a disease strongly associated with cognitive impairment, is on the rise globally with expectations that the number of patients will double every 20 years [35]. Early prevention of dementia is critical because no definitive therapy has been established. One of the promising sources for the prevention and treatment of different types of dementia, incl. Alzheimer's disease (AD), are plant sources [36]. In addition to evoking an antioxidant response, H. italicum essential oil also displays neuroprotective effects on mental fatigue/burnout, which generates further interest in Helichrysum extracts as potential cognitive-enhancing agents. Based on previous investigations, the H. italicum methanol-aqueous extract is worth investigating for the memory-ameliorating effects. We hypothesized that the combination of the H. italicum extract with the classical AChE inhibitor galantamine would support an improvement of the learning and memory process in the behavioral test in mice. To consider this hypothesis, we used a passive avoidance test to investigate whether 12 days of per oral administration of the combination lead to an improvement of the memory processes in comparison to the control group, single drug, or plant extract application. Such a combination could be used in the future for the development of specific cognitive-enhancing formulations. The incidence of dementia, a disease strongly associated with cognitive impai is on the rise globally with expectations that the number of patients will double ev years [35]. Early prevention of dementia is critical because no definitive therapy ha established. One of the promising sources for the prevention and treatment of di types of dementia, incl. Alzheimer s disease (AD), are plant sources [36]. In addi evoking an antioxidant response, H. italicum essential oil also displays neuropro effects on mental fatigue/burnout, which generates further interest in Helichrysum e as potential cognitive-enhancing agents. Based on previous investigations, the H. i methanol-aqueous extract is worth investigating for the memory-ameliorating effe hypothesized that the combination of the H. italicum extract with the classical AC hibitor galantamine would support an improvement of the learning and memory p in the behavioral test in mice. To consider this hypothesis, we used a passive avo test to investigate whether 12 days of per oral administration of the combination an improvement of the memory processes in comparison to the control group, singl or plant extract application. Such a combination could be used in the future for the opment of specific cognitive-enhancing formulations.
When the mice were treated with the combination of galantamine (3 mg/kg) italicum extract (200 mg/kg), a significant difference between combined and single cations was observed. It is worth noting that the combination was more beneficial memory-enhancement process in comparison with G. biloba (EGb 761) extract (p ≤ (Figure 6).
Taking into consideration that the antioxidant response is a key point in the me ameliorating effects, we proved the antioxidant activity of the H. italicum extract by When the mice were treated with the combination of galantamine (3 mg/kg) and H. italicum extract (200 mg/kg), a significant difference between combined and single applications was observed. It is worth noting that the combination was more beneficial for the memory-enhancement process in comparison with G. biloba (EGb 761) extract (p ≤ 0.001) ( Figure 6).
Taking into consideration that the antioxidant response is a key point in the memoryameliorating effects, we proved the antioxidant activity of the H. italicum extract by chemical-based assays based on the scavenging activity toward a stable free radical (DPPH and ABTS), the reduction of metal ions (FRAP and CUPRAC), metal chelating, and total antioxidant potential [37]. The main mechanism by which antioxidants play their protective role included hydrogen atom transfer (HAT) or a single electron transfer (SET). Often, more complex reactions like mixed HAT/SET, stepwise electron transfer-proton transfer, concerted electron-proton transfer, or sequential proton loss electron transfer occur [38]. The studied H. italicum extract demonstrated strong radical scavenging, metal-reducing, and chelating activity. Taking into account that the DPPH scavenging activity depends on hydrogen atom transfer (HAT); the phenolic compounds (hydroxycinnamic and acylquinic acids, and flavonoids) are very active due to the lower bond dissociation energies (BDE) of the phenolic hydroxyl groups [39]. Consistent with already published studies, we confirm a positive correlation between these phenolic compounds' classes in H. italicum and the antioxidant potential [15]. On the other hand, H. italicum extract demonstrated a moderate inhibitory activity towards acetylcholinesterase.
In line with the aforementioned results, we suggest that the main phenolic compounds belonging to hydroxycinnamic caffeoylquinic and acylhexaric acids, phloroglucinol derivatives, and some flavonoids (luteolin, quercetin, pinocembrin, naringenin) in H. italicum may hold significance for the memory enhancement observed in the passive avoidance test in mice.
Previously, caffeic, coumaric, and sinapic acids have been reported to improve cognitive function [40][41][42]. In different studies, the listed metabolites have been shown to suppress the breakdown of the amyloid-precursor protein, which is pathologically related to the Aβ (1-42) protein, lipid peroxidation, and neurite extension of hippocampal neurons [41,42]. These findings suggest that hydroxycinnamic acid intake may account for the improvement of the cognitive function, but the exact mechanism by which this intake affects cognitive function in humans is currently unknown. In the study of Kato et al. [43] on a small group of community-dwelling elderly individuals with complaints of subjective memory loss, after a 6-month intake period of caffeoylquinic acid there was significant improvement in attentional, executive, and memory functions.
Studies using mice and cultured neurons have shown that chlorogenic acid protect neurons and suppress the aggregation of amyloid beta (Aβ-one of the main hallmarks of AD) through antioxidant effects [41,44]. In addition, there are reports on beneficial effects of chlorogenic acid and/or its derivatives for ameliorating of spatial learning and memory and reducing behavioral deficits in a variety of in vivo animal models of disease or behavior [45].
Concerning flavones and flavonols, luteolin have been proved to exert a suppressive effect against endoplasmic reticulum stress activation and inflammatory signaling pathways in AD animal and cell models. Thus, the compound alleviates the learning and memory impairment in mice [46]. Numerous studies investigated the neuroprotective effects of quercetin in the central nervous system, especially in multiple in vitro and in vivo models of AD. Nakagawa et al. [47] reviewed seven studies of animal experiments estimating the neuroprotective effect of quercetin, in which quercetin improved cognition and memory deficits in rodent animal models of AD. The possible protective mechanisms of quercetin mainly involved the inhibition on Aβ aggregation and tauopathy, the anti-oxidative and anti-inflammatory activity, and amelioration of mitochondrial dysfunction [48].
A previous investigation demonstrated that the flavanone pinocembrin can be used to treat diseases such as stroke, AD, and vascular dementia [49,50]. Kang et al. revealed that pinocembrin attenuated learning and memory deficits induced by vascular dementia, by inducing the expression of Reelin, apoER2, and p-dab1 in the hippocampus. These authors also demonstrated that pinocembrin improved the impaired learning ability in rats by reducing the number of errors and decreasing the latency to step down in the step-down type of passive avoidance test [51]. Moreover, another flavanone naringenin dose dependently improved spatial recognition memory in Y maze, the discrimination ratio in a novel object discrimination task, and retention and recall capabilities in a passive avoidance test in the lipopolysaccharide-induced cognitive decline in rats. The authors suggested that naringenin have improved retention and recall in passive avoidance test via affecting synaptic plasticity [52]. Naringenin could ameliorate learning and memory deficit in passive avoidance tests in neurotoxic conditions through improvement of hippocampal oxidative stress and neuronal injury and increase the expression level of choline acetyltransferase [53].
It is worth noting that prenylated phloroglucinol α-pyrones and acylphloroglucinols could also attribute to the memory enhancing potential of H. italicum extract. It has been found that phloroglucinol reduced oxidative stress induced by oligomeric  in the HT-22 hippocampal cell line. In addition, the reduction in dendritic spine density caused by either hydrogen peroxide or Aβ1-42 was significantly rescued by phloroglucinol in rat primary hippocampal neuron cultures. Furthermore, phloroglucinol attenuated memory deficits in the 5XFAD mouse model of AD based on the Morris water maze and T-maze tests. As a whole, phloroglucinol displays a therapeutic potential for AD patients as a ROS-scavenger [54].
Herein, the most prominent effect of memory was found in the group treated with H. italicum extract combined with galantamine. In our previous study, galantamine successfully reverses scopolamine-induced memory impairment in mice, especially on the 12th day [55]. A characteristic that makes galantamine appropriate for the treatment of AD is a selective inhibitory activity on the enzyme acetylcholinesterase (AChE) in the central nervous system with a small effect on peripheral tissues [56]. Subsequently to galantamine's approval for the treatment of mild-to-moderate AD in 2001, a wide variety of species have been assessed in pursuit of new AChE inhibitors [57]. Gonçalves et al. [58] reported that the methanol extract of H. italicum, rich with phenolic compounds (caffeoylquinic and dicaffeoylquinic acids, and pinocembrin), showed high inhibitory activity against enzymes involved in Alzheimer's disease like AChE, tyrosinase, and α-glucosidase.
Our results confirm the well-known positive effect of G. biloba extract on the memory processes. After 12 days of administration, the group treated with G. biloba (EGb 761) statistically significantly prolonged the latency time in the passive avoidance test in comparison to the control group. A standardized extract of G. biloba leaves EGb 761 is a popular dietary supplement taken to enhance mental focus and used for treatment of certain cerebral dysfunctions and dementias associated with aging and AD [59].
The extract EGb 761 is known to contain about 24% flavonoids and 6% terpene lactones. There is reliable evidence that standardized Ginkgo extract exhibits several molecular and cellular neuroprotective mechanisms, including attenuation of apoptosis, inhibition of membrane lipid peroxidation, anti-inflammatory effects, and direct inhibition of Aβ aggregation. There is also data showing that G. biloba extract significantly inhibits the activity of AChE in the brain [60]. The positive effects of EGb 761 on memory function, including in stress situations, have been demonstrated in many experiments involving mice, rats, and even chicks while using various tests and paradigms, including conventional passive and active avoidance, Morris water maze, scopolamine-induced amnesia, learned helplessness, and olfactory learning ability, etc. These studies involve acute or chronic treatment with EGb and highlight the positive effect concerns on short-or long-term memory [61].
Poland, and kindly supplied by Prof. Dr. Adam Matkowski. A voucher specimen was deposited at Herbarium Academiae Scientiarum Bulgariae (SOM 178 491). Subsequently, the plant material was dried at room temperature.

Sample Extraction
Air-dried powdered aerial parts (50 g) were extracted with 80% MeOH (1:20 w/v) by sonication (100 kHz, ultra-sound bath Biobase UC-20C) for 15 min (×2) at room temperature. Then, the methanol was evaporated in vacuo and water residues were lyophilized (lyophilizer Biobase BK-FD10P) to yield crude extract 1.38 g. Afterwards, the lyophilized extract was dissolved in 80% methanol (0.1 mg/mL), filtered through a 0.45 µm syringe filter (Polypure II, Alltech, Lokeren, Belgium), and an aliquot (2 mL) of each solution was subjected to UHPLC-HRMS analyses. The same extract was used for further in vitro and in vivo tests.

UHPLC-HRMS Profiling
The phytochemical analyses were performed on a Q Exactive Plus mass spectrometer (ThermoFisher Scientific, Inc. Walthham, USA). The apparatus operated in negative and positive modes in m/z range from 100 to 1000. The chromatographic separation was performed on a reversed phase column Kromasil EternityXT C18 (1.8 µm, 2.1 × 100 mm) at 40 • C. The chromatographic analyses were performed as previously described [14]. Separation was achieved on an UHPLC system Dionex Ultimate 3000RSLC (ThermoFisher Scientific, Inc.) The mobile phase consisted of A: water (with 0.1% formic acid) and B: acetonitrile (with 0.1% formic acid). The used gradient was as follows: 5% B for 1 min, gradually turned to 30% B over 19 min, increased gradually to 50% B over 5 min, increased gradually to 70% B over 5 min, and finally increased gradually to 95% over 3 min. The system was then turned to the initial condition and equilibrated over 4 min. The flow rate was 300 µL/min and the injection volume was 1 µL. Data acquisition and processing were performed with Xcalibur 4.2 software (ThermoScientific, Walthham, USA). Peaks annotations were based on accurate masses in full MS and ddMS2, MS/MS fragmentation pathways, precursor and fragment ions relative abundance, elemental composition, comparison with the retention times, fragment spectra, and chromatographic behavior of reference standards obtained from an in-house database of previously identified compounds. The compounds identified with reference standards during the present study belong to confidence class 1, while the compounds that were putatively annotated belong to level 2 (reported in H. italicum previously), and putatively characterized classes belong to level 3 [29].
MZmine 2 software was applied to the UHPLC-HRMS raw files of the studied H. italicum extracts for the semi-quantitative analysis. Results are expressed as the % peak area of the compound to the total peak areas of the corresponding group secondary metabolites and all metabolites.

Antioxidant and Enzyme Inhibitory Assays
DPPH radical scavenging assay: The dsample solution (1 mg/mL; 1 mL) was added to 4 mL of a 0.004% methanol solution of DPPH. The sample absorbance was measured at 517 nm after a 30 min incubation [62].
ABTS radical scavenging assay: Briefly, ABTS+ was produced by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate and allowing the mixture to stand for 12-16 h in the dark at room temperature. The ABTS solution was diluted with methanol to an absorbance of 0.700 ± 0.02 at 734 nm. The sample solution (1 mg/mL; 1 mL) was added to the ABTS solution (2 mL) and mixed. The sample absorbance was measured at 734 nm after a 30 min incubation at room temperature [62].
CUPRAC assay: The sample solution (1 mg/mL; 0.5 mL) was added to the reaction mixture containing CuCl 2 (1 mL, 10 mM), neocuproine (1 mL, 7.5 mM), and NH 4 Ac buffer (1 mL, 1 M, pH 7.0). Similarly, a blank was prepared without CuCl 2 . Then, the sample and blank absorbances were measured at 450 nm after a 30 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample [62].
Metal chelating activity assay: Briefly, the sample solution (1 mg/mL; 2 mL) was added to FeCl2 solution (0.05 mL, 2 mM). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL). Similarly, a blank was prepared without ferrozine. Then, the sample and blank absorbances were measured at 562 nm after 10 min incubation at room temperature. The metal chelating activity was expressed as milligrams of EDTA (disodium edetate) equivalents (mg EDTAE/g extract) [62].
Phosphomolybdenum method: The sample solution (1 mg/mL; 0.3 mL) was mixed with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The sample absorbance was measured at 695 nm after a 90 min incubation at 95 • C. The total antioxidant capacity was expressed as millimoles of trolox equivalents (mmol TE/g extract) [62].

Animals
The experiment was conducted on 30 male mice, line H, with a body weight in the range of 28-32 g. All experiments were approved by the Institutional Animal Care Committee at the Medical University of Sofia. The animals were divided into 5 groups (n = 6). The tested substances were administered perorally (p.o.) in the following doses: Group 1: control group, receiving distilled water only; Group 2: animals treated with H. italicum extract-200 mg/kg; Group 3: animals treated with galantamine-3 mg/kg; Group 4: animals treated with Ginkgo biloba (EGB761)-100 mg/kg; Group 5: animals treated with galantamine (3 mg/kg) and H. italicum extract-200 mg/kg. The substances were in a solid form; they were ground, and the required amount of distilled water was added.
All treatments were performed for 12 days. During this period, animals were observed daily for behavioral changes and signs of toxicity. The experiment was conducted in accordance with the Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes (No. 346 of 28 February 2023) from the Bulgarian Food Safety Agency.

Passive Avoidance Test
The learning and memory processes of the experimental mice were evaluated by a passive avoidance test using an automated shuttle box (Gemini Avoidance System, San Diego Instruments). The apparatus consists of two identical compartments (25 × 20 × 16 cm each) with an electrified grid floor. The chambers are separated by a wall with a guillotine door (8 × 6 cm). The test started with an acclimatization period of 20 s. The maximum duration of the trial was 300 s. If a mouse entered the dark chamber, a weak electric shock with an intensity of 0.5 mA and a duration of 3 s. was applied through the grid floor. Learning was assessed by the latency of entry into the dark compartment. An increase in latency time indicated an improvement of the learning and memory processes. The tested compounds were administered p.o. 1 h before passive avoidance testing on days 1-5 of the experiment and then daily for 7 days without testing in the apparatus. On the 12th day the memory storage, processes were evaluated again by passive avoidance test.

Statistical Analysis
The results from the passive avoidance test were processed statistically using Graph-Pad Prism 6 software and presented as mean ± SEM. The groups were compared using one-way ANOVA followed by a post-hoc comparison of sample means (Tukey test). The differences were considered statistically significant at p ≤ 0.05.

Conclusions
For the first time, more than 90 secondary metabolites were reported in H. italicum including phenylethanoid glycosides, a series of hydroxybenzoic and hydroxicinnamic acid-glycosides, caffeoyl-hydroxydihydrocaffeoylquinic, p-coumaroyl-caffeoylquinic and tricaffeoylquinic acids, malonyl-dicaffeoylquinic acids, a series of caffeoylhexaric acids, and methoxylated derivatives of scutelarein, quercetagetin, and 6-hydroxyluteolin. The main finding of the phytochemical study was a comprehensive profiling of heterodimer-pyrones where 23 compounds, undescribed in the literature, were annotated. This study is the first attempt to propose the fragmentation patterns of four subclasses pyrones in LC-HRMS. For the first time, the cognitive-enhancing properties of H. italicum extract is reported. We have demonstrated that the combination of the extract and classical acetylcholinesterase inhibitor galantamine significantly improved the learning and memory after 12 days administration in a passive avoidance test in mice. The effect is more pronounced even than that of G. biloba (EGb 761). Taken together, our data suggest significant antioxidant activity of H. italicum extract by radical scavenging activity, reducing power and metal chelating capacity. The extract showed moderate acetylcholinesterase and low butyrylcholinesterase inhibitory potential. It appears that powerful antioxidant activity coupled with moderate and selective AChE inhibitory ability accounted for the cognitive-enhancing potential of H. italicum extract. Prenylated phloroglucinol derivatives, acylquinic and acylhexaric acids, and flavonoids may hold significance for the memory improvement potential of H. italicum extract. Further analysis concerning mechanisms of action is needed to advance our knowledge on the pharmacological effects of H. italicum.